Single-minded, Dmef2, Pointed, and Su(H) act on identified regulatory sequences of the roughest gene in Drosophila melanogaster.

Roughest (Rst) is a cell adhesion molecule of the immunoglobulin superfamily that has multiple and diverse functions during the development of Drosophila melanogaster. The pleiotropic action of Rst is reflected by its complex and dynamic expression during the development of Drosophila. By an enhancer detection screen, we previously identified several cis-regulatory modules that mediate specific expression of the roughest gene in Drosophila developmental processes. To identify trans-regulators of rst expression, we used the Gal4/UAS system to screen for factors that were sufficient to activate Rst expression when ectopically expressed. By this method we identified the transcription factors Single-minded, Pointed.P1, and Su(H)-VP16. Furthermore, we showed that these factors and, in addition, Dmef2 are able to ectopically activate rst expression via the previously described rst cis-regulatory modules. This fact and the use of mutant analysis allocates the action of the transcription factors to specific developmental contexts. In the case of Sim, we could show that it regulates rst expression in the embryonic midline, but not in the optic lobes. Mutagenesis of Sim consensus binding sites in the regulatory module required for rst expression in the embryonic midline, abolished rst expression; indicating that the regulation of rst by Sim is direct.

Interspecies comparison of a gene pair with partially redundant function: the rst and kirre genes in D. virilis and D. melanogaster.

The D. melanogaster rst and kirre genes encode two highly related immunoglobulin-like cell adhesion molecules that function redundantly during embryonic muscle development. The two genes appear to be derived from a common ancestor by gene duplication. Gene duplications have been proposed to be of major evolutionary significance since duplicated redundant sequences can accumulate mutations without detrimental effects for the organism and leave the duplicated genes free to assume novel functions. To address the issue of conservation of the duplicated sequences and their putative redundancy, as well as to identify putative functional divergence of the paralogs during drosophilid evolution, we performed an interspecies comparison of the rst and kirre genes from D. virilis and D. melanogaster. The D. virilis genome contains orthologues of both rst and kirre and hence the duplication took place before the split of the two lineages and has subsequently been conserved. However, whilst the Rst orthologues show a high degree of sequence similarity, this similarity is lower in Kirre orthologues. Especially the intracellular domains of D. virilis and D. melanogaster Kirre sequences are highly divergent: the D. virilis kirre gene lacks the 3'-most exon present in D. melanogaster, which contains motifs conserved between kirre and rst in D. melanogaster. Hence, while each of the two genes is highly conserved at the level of its exon-intron organization, the selection forces acting on the rst and kirre coding sequences are different. These findings are discussed in the light of general evolutionary mechanisms.